ADP-ribosylation factors (ARFs) are -20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activities of cholera toxin in vitro and function in protein trafficking through the ER-Golgi system in vivo. One membrane (mARF) and two cytosolic (sARFI and sARFII) forms have been purified. ARF-immunoreactive material is widespread but predominantly found in neural tissue. It has been observed in every eucaryotic species examined from Giardia to human but was not detected in E. coli. Six mammalian ARFs cloned from bovine and/or human cDNA libraries can be grouped in three classes based on size and amino acid sequence identify. ARF 2 (Class I) is encoded by a -2.6-kb mRNA that is developmentally regulated in rat brain. However, unlike ARFs 1 and 3 to 6, whose mRNAs are conserved across species, ARF 2 mRNA, although present in other species, was not detected in poly(A)+ RNA from monkey and human. Further, ARF 2 mRNA appears to exhibit selective tissue expression. To begin to elucidate the mechanism underlying the differential expression of ARF 2, a composite cDNA (-2270 bp minus the poly(A) tail) was constructed from a partial cDNA clone and sequence of PCR-amplified fragments from reverse-transcribed poly(A)+ RNA. In the 3'-untranslated region (UTR), there were two potential polyadenylation signals, and two ATTTA motifs, believed to signal mRNA degradation. The bovine ARF 2 gene, represented in three overlapping genomic clones, spanned -20 kbp with five exons and four introns. There were multiple transcriptional initiation sites; the promoter region lacked a TATA-like sequence, but contained six inverted CCAAT boxes, four potential Sp1 binding sites, and a potential AP- 2 binding site. Comparison of the bovine ARF 2 gene with the human ARF 1 and 3 genes indicates that the gene structures of the class I ARFs are conserved among its members and across species. However, unlike ARF 1 and 3 promoter regions, that of the ARF 2 gene is not highly G/C rich and contains an inverted CCAAT box in the correct position for regulating transcriptional activity.